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The Journal of Immunology, 1999, 163: 6702-6711.
Copyright © 1999 by The American Association of Immunologists

Kupffer Cells from Schistosoma mansoni-Infected Mice Participate in the Prompt Type 2 Differentiation of Hepatic T Cells in Response to Worm Antigens1

Nobuki Hayashi2,*, Kiyoshi Matsui*, Hiroko Tsutsui{dagger},||, Yoshio Osada, Raafat T. Mohamed, Hiroki Nakano{dagger}, Shin-ichiro Kashiwamura{ddagger}, Yasuko Hyodo*, Kiyoshi Takeda§,||, Shizuo Akira§,||, Toshikazu Hada*, Kazuya Higashino*,{ddagger}, Somei Kojima and Kenji Nakanishi3{dagger},{ddagger},||

* Third Department of Internal Medicine, {dagger} Department of Immunology and Medical Zoology, and {ddagger} Laboratory of Host Defenses Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; § Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan; Department of Parasitology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and || Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Infection with Schistosoma mansoni, a portal vein-residing helminth, is well known to generate life cycle-dependent, systemic immune responses in the host, type 1 deviation during the prepatent period, and type 2 polarization after oviposition. Here we investigated local immunological changes in the liver after infection. Unlike splenocytes, hepatic lymphocytes from infected mice during the prepatent period already produced a higher amount of IL-4 and a lesser amount of IFN-{gamma} than those from uninfected mice. Hepatic lymphocytes, particularly conventional T cells, but not NK1.1+ T cells, promptly produced IL-4 in response to worm products, soluble worm Ag preparation (SWAP), whenever presented by Kupffer cells from infected mice. The hepatic lymphocytes that had been stimulated with SWAP presented by infected mice-derived Kupffer cells produced a huge amount of IL-4, IL-13, and IL-5 as well as little IFN-{gamma} in response to immobilized anti-CD3 mAb. Kupffer cells from uninfected mice produced IL-6 and IL-10, but not IL-12 or IL-18, in response to SWAP stimulation and gained the potential to additionally produce IL-4 and IL-13 after the infection. These results suggested that prompt type 2 deviation in the liver after the infection might be due to the alteration of Kupffer cells that induces SWAP-mediated type 2-development of hepatic T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Macrophages are an essential component of both innate immunity and acquired immunity. Macrophages act as effector cells by their specialized lysosomal enzymes and cytotoxic molecules, such as nitric oxide, oxygen radicals, and TNF-{alpha} in both systems, and also play an important role as APCs in the acquired immune system (1, 2, 3, 4, 5). In either system, macrophages regulate and modify the immune reaction by secretion of various cytokines. IL-12 and IL-18, originally designated IFN-{gamma}-inducing factor, initiate and accelerate inflammatory reactions, including type 1 immune responses, respectively (6, 7, 8, 9, 10), whereas IL-10 down-regulates them (8). Particularly, IL-12 produced by macrophages is demonstrated to primarily initiate type 1 T cell differentiation in vitro and in vivo (6, 8, 9, 10, 11). This is also the case for tissue-localized immunity. As previously shown, administration of heat-killed Propionibacterium acnes (3) directs hepatic immune competent cells to type 1 shift, which is induced and augmented by IL-12 and IL-18 produced by P. acnes-elicited Kupffer cells, a tissue type of macrophages in the liver (12, 13).

Infection with Schistosoma mansoni results in hepatic fibrosis and occasionally lethal liver cirrhosis in hosts, including humans and mice (14, 15). Infection with S. mansoni also induces proper and unique changes in the host immune system according to the stage of its life cycle. After infection with cercariae of S. mansoni through the skin, larvae transform to schistosomula, migrate into the lungs, and eventually reach the intrahepatic portal circulation. Within several weeks, the worms mate and migrate to the mesenteric veins, and female worms lay eggs. Many investigators have reported that splenic T cells from S. mansoni-infected mice show life cycle-dependent changes in cytokine production profiles, in that they become type 1 T cells during the prepatent period and change to type 2 T cells after the beginning of egg deposition (16). Type 2 deviation in the spleen has been reported to be in part attributable to host cell reactions to soluble egg Ag (SEA)4 (17, 18). In this study we investigated whether the hepatic immune system shows a reaction to S. mansoni-infection similar to that in the spleen, particularly focusing on the role of Kupffer cells in the immunological reactions. We found that hepatic T cells deviated into type 2 T cells without showing any type 1 shift. This was already observed in mice during the prepatent period. To investigate the mechanism of how the hepatic immune system promptly shifts to type 2, we analyzed the responses of hepatic immune competent cells to adolescent worm products, soluble worm Ag preparation (SWAP) (19). Kupffer cells from uninfected mice produced IL-10 and IL-6, but do not produce IL-12 or IL-18 in response to SWAP. Moreover, Kupffer cells prepared from S. mansoni-infected mice produced IL-4 and IL-13. Hepatic lymphocytes produced IL-4, but not IFN-{gamma}, in response to SWAP whenever presented by Kupffer cells from S. mansoni-infected mice. We show that these unique properties of SWAP, Kupffer cells, and hepatic lymphocytes contribute to the prompt and accelerated type 2 response in the liver following infection with S. mansoni.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Infection with S. mansoni

Female C57BL/6 mice (5–7 wk old) from Japan SLC (Sizuoka, Japan) were infected with 40 cercariae of S. mansoni via tail skin. STAT6-deficient mice with C57BL/6 background (female, 5–7 wk old) were used (20, 21).

Reagents

SWAP was prepared from homogenized adult worms as described by Pearce et al. with some modification (19). Briefly, fat was removed from adult worms by homogenizing them in cold diethyl ether, and nonfat pellet was suspended in veronal buffered saline. Adult worm extract was prepared by freezing and thawing of the pellet suspension, dialyzed against PBS, filtered, and stored at -80°C until use as SWAP. Before use for cell stimulation, SWAP was preincubated with 100 U/ml polymyxin B for 1 h at room temperature to neutralize possibly contaminated LPS (22, 23). Anti-IL-18 mAbs used for ELISA for mouse IL-18 were provided by Hayashibara Biochemical Laboratories (Okayama, Japan) (12, 24). 2C11 (directed against CD3{epsilon} chain, hamster IgG) or FITC-conjugated 2C11, FITC-conjugated anti-CD11b mAb (Mac-1 {alpha}-chain, M1/70, rat IgG2b), FITC-conjugated anti-CD45R mAb (B220, RA3-6B2, rat IgG2a), FITC-conjugated anti-mouse IgM mAb (R6-60.2, rat IgG2a), PE-conjugated anti-CD117 mAb (c-Kit, 2B8, rat IgG2b), biotinylated anti-NK1.1 mAb (PK136, rat IgG2a), FITC-conjugated anti-CD4 mAb (RM4–5, rat IgG2a), and anti-Fc{gamma}R II/III mAb (2.4G2, rat IgG2b) were purchased from PharMingen (San Diego, CA). PE-streptavidin was obtained from Becton Dickinson (Mountain View, CA). Biotinylated anti-IL-4 mAb (BVD6-24G2), and PE-conjugated anti-IL-4 mAb (11B11) were purchased from PharMingen. Anti-Thy-1.2 mAb (HO-13-4) was provided by Dr. W. E. Paul, National Institutes of Health (Bethesda, MD). Guinea pig complement was purchased from Cedarlane (Hornby, Canada). Endotoxin-free, neutralizing anti-IL-6 mAb (MP5-20F3), anti-IL-10 mAb (JES5-2A5), anti-B7.1 mAb (16-10A1), and anti-B7.2 mAb (PO3.1) were purchased from PharMingen. Hybridoma producing neutralizing anti-IL-4 mAb (11B11) from American Type Culture Collection (Manassas, VA) was inoculated in the abdomen of BALB/c-nu/nu (SLC), and purified Ig from the ascites was used for the neutralization experiments. The culture medium generally used in this study was RPMI 1640 containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-ME, and 2 mM L-glutamine. All experiments were performed in triplicate. All data are given as the mean ± SEM.

Cell preparation

Splenic T cells were prepared from C57BL/6 mice before or 4 or 16 wk after infection with S. mansoni by passing them through a nylon wool column. Spleen cells from variously treated mice were incubated in plastic dishes for 1 h at 37°C, and adherent cells were additionally incubated in fresh plastic dishes for another 1 h. The adherent cells were used as splenic macrophages.

Liver lymphocytes were prepared from STAT6-deficient mice or C57BL/6 mice before or 2, 3, 4, 10, or 16 wk after infection with S. mansoni cercariae as described previously (12). Kupffer cells were prepared from C57BL/6 mice before or 3 or 10 wk after infection with S. mansoni by collagenase-Pronase digestion followed by elutriation centrifugation as described previously (25).

FACS analysis

Fluorescence staining of Kupffer cells or liver lymphocytes was performed after treatment with anti-Fc{gamma}R mAb (12). Kupffer cells from C57BL/6 mice infected with S. mansoni 10 wk previously were stained with FITC-conjugated anti-CD3 mAb, FITC-conjugated anti-11b mAb, FITC-conjugated anti-IgM mAb, FITC-conjugated anti-CD45R mAb, or PE-conjugated anti-CD117 mAb. Liver lymphocytes and splenic T cells from variously infected C57BL/6 mice or STAT6-deficient mice were stained with biotinylated anti-NK1.1 followed by incubation with FITC-anti-CD3 or FITC-anti-CD4 and PE-streptavidin. Stained cells were analyzed using a dual laser FACScalibur (Becton Dickinson). Ten thousand cells were analyzed for each assay, and data were processed with CellQuest (Becton Dickinson).

For the indicated experiments, Kupffer cells (1 x 106/ml) from mice infected 10 wk previously were incubated in a 24-well plate with 200 µg/ml of SWAP for 6 h, during the last 30 min of which the cells were incubated additionally with a 1/1000 volume of FluoSpheres (carboxylate-modified microspheres labeled with yellow-green fluorescence (Molecular Probes, Eugene, OR)). The plate was vigorously washed to remove free beads, and the cells collected were processed for intracellular staining with PE-conjugated anti-IL-4 mAb (PharMingen), as shown previously (26).

Immunohistochemistry

Kupffer cells (1 x 106/ml) from mice infected for 10 wk were incubated in a 24-well plate with 200 µg/ml of SWAP for 18 h, during the last 30 min of which the cells were additionally incubated with a 1/1000 volume of FluoSpheres. Immunohistochemistry was performed using 10 µg/ml of biotinylated anti-IL-4 mAb followed by ABC kits (Vector, Burlingame, CA), as previously reported (27).

T cell depletion

Liver lymphocytes from C57BL/6 mice before or 3 wk after infection with S. mansoni were treated with two rounds of complement-mediated lysis of T cells with monoclonal anti-Thy-1.2 mAb (28). This procedure routinely yields cells that are <2% CD3 positive.

Cell culture

Liver lymphocytes or splenic T cells (2 x 105/well/200 µl) were incubated on anti-CD3-coated 96-well plates for 24 h (12). Kupffer cells or splenic macrophages from variously treated C57BL/6 mice (2 x 106/ml) were incubated with SWAP (200 µg/ml) or LPS (Detroit, Detroit, MI; 1 µg/ml) in 24-well plates for 24 h.

Liver lymphocytes or splenic T cells (5 x 105/ml) from variously treated C57BL/6 mice or uninfected STAT6-deficient mice were incubated with 1 x 106/ml of Kupffer cells or splenic macrophages from uninfected C57BL/6 mice or infected C57BL/6 mice in the presence of 200 µg/ml of SWAP in a 24-well plate for 24 h. For the indicated experiments, T cell-depleted hepatic lymphocytes (5 x 105/ml) from uninfected C57BL/6 mice were incubated with 200 µg/ml of SWAP in the presence of Kupffer cells (1 x 106/ml) from variously treated mice for 24 h.

Secondary culture

Liver lymphocytes from uninfected mice (5 x 105) were incubated for 24 h with 200 µg/ml of SWAP in the presence of Kupffer cells (1 x 106) from mice infected with S. mansoni 10 wk previously, and nonadherent cells (5 x 105/ml) were vigorously washed with PBS and recultured on anti-CD3 mAb-coated plates for another 24 h. For the control study, the freshly isolated liver lymphocytes were incubated on an anti-CD3 mAb-coated plate for 24 h for detection of various kinds of cytokines. For the indicated experiments, the liver lymphocytes from uninfected mice (5 x 105) were incubated with 200 µg/ml of SWAP and the infected mouse-derived Kupffer cells (1 x 106) in the presence of anti-IL-4 mAb (100 µg/ml), anti-IL-10 mAb (10 µg/ml), anti-IL-6 mAb (10 µg/ml), anti-B7.1 mAb (20 µg/ml), or anti-B7.2 mAb (20 µg/ml) for 24 h. Nonadherent lymphocytes were collected, vigorously washed with PBS, and incubated on fresh anti-CD3 mAb-coated plates for another 24 h.

Assay for cytokines

The concentrations of cytokines were determined by ELISA. IL-4 and IFN-{gamma} were determined by ELISA kits from Genzyme (Boston, MA). IL-12 p40, IL-10, TNF-{alpha}, and IL-6 levels were measured by ELISA kits from BioSource (Camarillo, MA). The IL-18 level was determined by ELISA as shown previously (12). IL-5 and IL-13 levels were also measured by ELISA kits from Endogen (Woburn, MA) and R & D (Minneapolis, MN), respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Direct type 2 shift in the liver of S. mansoni-infected mice

Since S. mansoni resides only in the hepatic portal vein after attaining the adolescent stage 3–4 wk after the infection (14), we assumed that the hepatic immune system shows a unique local reaction to S. mansoni. We examined chronological changes in cytokine production profile of hepatic T cells after S. mansoni infection compared with those of splenic T cells. As shown in Fig. 1Go, like splenic T cells, hepatic T cells prepared from uninfected mice produced both IL-4 and IFN-{gamma} in response to immobilized anti-CD3 mAb. As expected (16), splenic T cells showed type 1 shift at the adolescent worm stage and at 4 wk after infection, and then turned into type 2 T cells after S. mansoni began to lay eggs at 8 wk and later after the infection (Fig. 1Goa, upper panel). In contrast, hepatic T cells already showed type 2 shift at the adolescent worm stage, and their type 2 polarization was further facilitated after starting egg deposition (Fig. 1Goa, lower panel).



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  		FIGURE 1. Straightforward type 2 polarization in the liver after S. mansoni infection. a, Splenic T cells (Spleen) and hepatic lymphocytes (Liver) were isolated from mice at the indicated time points after the infection with S. mansoni. The lymphocytes were cultured on anti-CD3 mAb-coated 96-well microtiter plates for 24 h. IL-4 (hatched column) and IFN-{gamma} (closed column) in each resulting supernatant were measured by ELISA. IFN-{gamma} and IL-4 were not observed in the supernatant of splenic T cells or liver lymphocytes incubated in the absence of anti-CD3 mAb. The data are the mean ± SEM of triplicate samples from one experiment. b, The surface phenotypes of the cells prepared in a were determined by FACS after staining them with FITC-conjugated anti-CD4 mAb and biotinylated anti-NK1.1 mAb followed by PE-streptavidin. The results shown are representative of three independent experiments. ND, not detected.

 
To investigate whether the different cytokine profiles between spleen and hepatic T cells was due to the difference in the composition of the cell population, we analyzed cell proportions, particularly focusing on that of CD4+NK1.1+ T cells because of their prompt production of IL-4, an essential cytokine to differentiate naive T cells into type 2 T cells directly (29, 30, 31, 32). As shown in Fig. 1Gob, the proportion of CD4+NK1.1+ T cells increased in splenic T cells after infection. By contrast, the proportion of this population was continuously reduced in liver after infection. The cell yield of hepatic lymphocytes from infected mice was, at most, about 1.5-fold more than that from uninfected mice (data not shown). These differences and changes in composition of both populations before and after the infection did not allow us to determine whether different cytokine profiles of splenic and hepatic T cells reflected their different cell compositions or their functional capacities.

A more detailed kinetic study revealed an exponential type 2 polarization of hepatic lymphocytes with down-regulated IFN-{gamma} production after the infection (Fig. 2Go). Histological study disclosed that eosinophils accumulated around the worm in liver of mice at 4 wk postinfection, giving another clue to early type 2 shift in liver after infection (data not shown). Thus, hepatic T cells directly and promptly developed into type 2 T cells without showing a remarkable type 1 shift after S. mansoni infection.



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  		FIGURE 2. Type 2 dominant immune responses in the liver after S. mansoni infection. Liver lymphocytes from uninfected or S. mansoni-infected mice at the indicated time points after infection were incubated with immobilized anti-CD3 mAb for 24 h. Culture supernatants were harvested and tested for their concentrations of IL-4 (hatched column) and IFN-{gamma} (closed column). The data are the mean ± SEM of triplicate samples from one experiment. The results shown are representative of three independent experiments. ND, not detected.

 
Unique cytokine production profile of SWAP-stimulated Kupffer cells

Since hepatic immune system shows a unique type 2 immune response upon S. mansoni infection at adolescent worm stage (Figs. 1Go and 2Go), we investigated the regulatory role of Kupffer cells in this type 2 deviation by examining their cytokine production profile in response to stimulation with SWAP compared with that to LPS, a potent, bacteria-derived stimulus for macrophages (25). As shown in Table IGo, Kupffer cells from uninfected mice produced almost the same level of IL-10 in response to both stimuli, but they produced 8-fold more IL-6 in response to LPS than in response to SWAP (33, 34). In contrast, the same cells produced a much lesser amount of type 1 driving cytokines, IL-12 and IL-18 (7, 8, 9, 11, 24), in response to SWAP stimulation than in response to LPS. Thus, SWAP appears to stimulate Kupffer cells to fail to induce type 1 immune responses.


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  		Table I. Comparison of cytokine production profiles between LPS- and SWAP-stimulated Kupffer cells1

 
IL-4 production by Kupffer cells from infected mice in response to SWAP

Next, we examined whether S. mansoni infection changes the cytokine production profile of Kupffer cells in response to SWAP. As shown in Fig. 3Goa, Kupffer cells from the mice in the prepatent period remained to produce IL-6 and IL-10 in response to stimulation with SWAP. Kupffer cells still failed to secrete TNF-{alpha}, IL-12, or IL-18 in response to SWAP (data not shown). To our surprise, at 3 wk after the infection they started to produce IL-4 in response to SWAP (Fig. 3Goa). As shown in Table IGo and Fig. 3Goa, the capacity for IL-4 production was tremendously increased as infection progressed to the egg deposit stage (describe below). To confirm that infected mouse-derived Kupffer cells can produce IL-4, we examined intracellular IL-4 staining of the Kupffer cell fraction. As shown in Fig. 3Gob, left, phagocytes in the cell preparation were able to be stained with anti-IL-4 mAb directly, indicating that Kupffer cells, at least hepatic adherent phagocytes, produced IL-4 in response to SWAP. The specificity of PE-conjugated mAb against IL-4 was proven by competitive inhibition by an excess of unconjugated mAb in a separate experiment. This was also proven by immunohistochemistry. As shown in Fig. 3Gob, right, Kupffer cells from infected mice that has a property common to monocytes-macrophages of large cytoplasm with relatively small nucleus, ingest beads and also have cytoplasm stained with anti-IL-4 mAb. The Kupffer cell fraction contained many cells similar to the cell shown in Fig. 3Gob, right. The cells were not stained when incubated with control Ab instead of anti-IL-4 mAb (data not shown). Moreover, the SWAP-stimulated Kupffer cells from the infected mice also produced IL-13, a second, potent, type 2-inducing cytokine (Table IGo) (35, 36). Interestingly, Kupffer cells from S. mansoni-infected mice (at 10 wk) did not lose the potential to produce IL-12, IL-18, and TNF-{alpha}, because Kupffer cells produced all the cytokines except IL-4 and IL-13 in response to LPS (Table IGo). Thus, Kupffer cells gained the capacity to produce IL-4 and IL-13 uniquely in response to adult worm products during infection.



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  		FIGURE 3. Cytokine production profile of SWAP-stimulated S. mansoni-infected Kupffer cells. a, Kupffer cells started to produce IL-4 after S. mansoni infection. Kupffer cells prepared from the uninfected mice (open column) or S. mansoni-infected mice at 3 wk (closed column) or 10 wk (hatched column) were incubated with or without 200 µg/ml of SWAP for 24 h. Culture supernatants were harvested and tested for their concentrations of IL-4, IL-6, and IL-10. ND, not detected. The data shown are the mean ± SEM of triplicate samples of one experiment. b, IL-4 production by Kupffer cells. Kupffer cells from infected mice at 10 wk were incubated with SWAP for 5.5 h and additionally with fluorescence-labeled beads for 30 min. The cells were then intracellularly stained with PE-conjugated anti-IL-4 mAb. The intensity of intracellular IL-4 and the level of phagocytosis were determined by FACS (left). SWAP-stimulated Kupffer cells were also immunohistochemically stained using biotinylated anti-IL-4 mAb followed by ABC kit (right). Kupffer cells ingested beads as indicated by arrowheads, and IL-4 was homogeneously detected in its cytoplasm. The photo is representative of three independent experiments with similar results. c, Lack of CD3+, CD45R+, IgM+, or c-Kit+ cells in the Kupffer cell fraction. Kupffer cells from S. mansoni-infected mice at 10 wk were stained with FITC-anti-CD3, FITC-anti-11b, FITC-anti-IgM, FITC- anti-CD45R, or PE-anti-CD117 after Fc{gamma}R blocking. The surface phenotypes were determined by FACS analysis. The results shown are representative of three independent experiments with similar results.

 
Next, we confirmed that the Kupffer cell fraction from infected mice was not contaminated with IL-4-producing cells, such as T cells and mast cells (37), by FACS analysis. As shown in Fig. 3Goc, the Kupffer cell fraction isolated from infected mice did not contain T cells, B cells, or c-Kit+ cells. Mac1+ cells in the cell preparation appeared to be about 70%. A similar staining pattern was observed in the Kupffer cell fraction from uninfected mice, which contained 95% Kupffer cells, as determined by phagocytosis of latex beads and by morphological property (12, 25).

Prompt IL-4 production by hepatic lymphocytes in response to SWAP presented by Kupffer cells from the infected mice during the prepatent period

Next, we investigated whether SWAP can stimulate hepatic T cells to produce IL-4 directly or with help from Kupffer cells. To test this, we prepared hepatic lymphocytes or splenic T cells from uninfected mice or mice infected with S. mansoni 3 wk previously, incubated them with SWAP together with Kupffer cells or splenic macrophages from uninfected or infected mice for 24 h, and measured IL-4 in each resulting supernatant. Infected mouse-derived Kupffer cells incubated with SWAP produced 80 pg/ml of IL-4 in the culture supernatant (Fig. 4Goa). Hepatic lymphocytes from uninfected or infected mice produced much more IL-4 in response to SWAP presented by Kupffer cells from infected mice than the Kupffer cells themselves produced in response to SWAP, indicating that hepatic lymphocytes can respond to SWAP presented by the appropriate APCs, infected mouse-derived Kupffer cells. The same hepatic lymphocytes, however, did not produce IL-4 in response to SWAP in the absence of the Kupffer cells from infected mice, indicating that hepatic lymphocytes absolutely require APCs to respond to SWAP (data not shown). The hepatic T cells did not produce IL-4 even in the presence of infected mouse-derived Kupffer cells whenever SWAP was not added to the culture (data not shown). Furthermore, hepatic lymphocytes from either group of mice did not produce IL-4 in response to SWAP stimulation in the presence of Kupffer cells from uninfected mice (Fig. 4Goa), indicating that only Kupffer cells from infected mice have the ability to present SWAP to hepatic lymphocytes. IFN-{gamma} was not detectable in resulting supernatants of any reconstitution mixture in response to SWAP (data not shown).



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  		FIGURE 4. IL-4 production by hepatic lymphocytes in response to SWAP presented by infected mouse-derived macrophages. a, Liver lymphocytes or splenocytes from uninfected mice (hatched column) or S. mansoni-infected mice at 3 wk (closed column) were incubated with Kupffer cells from uninfected mice or S. mansoni-infected mice at 3 wk in the presence or the absence of 200 µg/ml of SWAP for 24 h. IL-4 in each resulting supernatant was measured. b, Liver lymphocytes or splenocytes from uninfected mice (hatched column) or S. mansoni-infected mice at 3 wk (closed column) were incubated with splenic macrophages from uninfected mice or S. mansoni-infected mice at 3 wk in the presence or the absence of 200 µg/ml of SWAP for 24 h. IL-4 in each supernatant was measured. Dotted horizontal lines indicate the mean amount of IL-4 produced by infected mouse-derived macrophages in response to SWAP (Kupffer cells, 40 pg/ml; splenic macrophages, undetectable). The data are the mean ± SEM of triplicate samples from one experiment. The results shown are representative of three independent experiments with similar results. ND, not detected.

 
In contrast to hepatic lymphocytes, splenic lymphocytes from uninfected or infected mice did not produce IL-4 in response to SWAP, even when infected mouse-derived Kupffer cells were used as APCs (Fig. 4Goa). As shown in Fig. 4Gob, hepatic lymphocytes produced IL-4, but much less, in response to stimulation with SWAP in the presence of splenic macrophages isolated from infected mice than in that of Kupffer cells from the same infected mice. Unlike Kupffer cells, splenic macrophages from infected mice did not produce IL-4 in response to SWAP (Fig. 4Gob). These stimulation conditions also did not induce IFN-{gamma} production from either hepatic or splenic lymphocytes (data not shown). Thus, only hepatic lymphocytes showed potency to produce IL-4 in response to stimulation with SWAP, and Kupffer cells from the infected mice had a much higher capacity to present SWAP than splenic macrophages, presumably due in part to IL-4 production by themselves in response to SWAP; this will be examined later (Fig. 6Go).



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  		FIGURE 6. Prompt type 2 differentiation of hepatic T cells after stimulation with SWAP. a, SWAP presented by Kupffer cells induced type 2 differentiation in hepatic T cells. Hepatic lymphocytes from uninfected mice were first incubated for 24 h with SWAP in the presence of Kupffer cells from infected mice. After being vigorously washed, lymphocytes were incubated on anti-CD3 mAb-coated plates for another 24 h, and the concentration of each cytokine was measured (closed columns). For the control study, hepatic lymphocytes freshly isolated from the same uninfected mice were incubated on anti-CD3 mAb-coated plates for 24 h, and cytokine levels were determined (open columns). b, IL-4-independent type 2 differentiation of hepatic T cells by SWAP presented by Kupffer cells. Hepatic lymphocytes from uninfected mice were also incubated with SWAP in the presence ({alpha}-series) or the absence (Control) of various kinds of neutralizing mAbs as well as infected mouse-derived Kupffer cells for 24 h, and the secondary culture was performed according to the same protocol as that shown in a. Cytokines in each supernatant were measured by ELISA. The data are the mean ± SEM of triplicate samples from one experiment. The results shown are representative of three independent experiments with similar results. ND, not detected.

 
IL-4 production by conventional hepatic T cells in response to SWAP

We investigated what cell type in the liver produces IL-4 in response to SWAP presented by infected mouse-derived Kupffer cells. To test the contribution of T cells to this phenomenon, we depleted T cells from hepatic lymphocytes in vitro and incubated them under the same conditions as those described in Fig. 4Goa. As shown in Fig. 5Goa, both CD3+NK1.1- T cells and CD3+NK1.1+ T cells were eliminated after T cell depletion. Infected mouse-derived Kupffer cells produced 80 pg/ml of IL-4 in response to SWAP (Fig. 4Goa). Hepatic lymphocytes from infected or uninfected mice did not produce IL-4 in response to SWAP even in the presence of Kupffer cells, once T cells were depleted (Fig. 5Gob). They did not produce IFN-{gamma} under any stimulation condition (data not shown). Thus, T cells of hepatic lymphocytes have the potential to produce IL-4 in response to SWAP presented by Kupffer cells from infected mice.



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  		FIGURE 5. Conventional T cells promptly produced IL-4 in response to SWAP presented by infected mouse-derived Kupffer cells. a, T cell depletion from hepatic lymphocytes. T cells were depleted from liver lymphocytes from uninfected mice or S. mansoni-infected mice at 3 wk using anti-Thy-1.2 mAb followed by incubation with complement for two cycles. The effectiveness of depletion was monitored by FACS analysis of cells stained with biotinylated anti-NK1.1 mAb followed by PE-streptavidin and FITC-conjugated anti-CD3 mAb. The results shown are representative of three independent experiments with similar results. b, Lack of IL-4 production by T cell-depleted hepatic lymphocytes in response to SWAP. Untreated (Whole) or T cell-depleted (T depleted) hepatic lymphocytes from uninfected mice (hatched column) or infected mice at 3 wk (closed column) were incubated with Kupffer cells prepared from mice infected with S. mansoni at 3 wk in the presence of SWAP for 24 h to determine the IL-4 response. c, Surface phenotypes of hepatic lymphocytes from STAT6-deficient mice similar to those from wild-type mice. Hepatic lymphocytes from uninfected STAT6-deficient mice or wild-type mice (C57BL/6) were stained with biotinylated anti-CD3 mAb followed by PE-streptavidin and FITC-conjugated anti-NK1.1 mAb. d, Impaired IL-4 production by hepatic lymphocytes from STAT6-deficient mice in response to SWAP. Hepatic lymphocytes from uninfected STAT6-deficient mice (hatched column), uninfected wild-type mice (hatched column), or S. mansoni-infected wild-type mice at 3 wk (closed column) were incubated under the conditions described in b. Dotted horizontal lines in b and d indicate the mean amount of IL-4 produced by infected mouse-derived Kupffer cells in response to SWAP. The data are the mean ± SEM of triplicate samples from one experiment. The results shown are representative of three independent experiments. ND, not detected.

 
Since the liver has many more NK1.1+ T cells, particularly NK1.1+CD4+ T cells, than other tissues (12, 29), we investigated which cell type of hepatic T cells, NK1.1+ T cells and/or NK1.1- T cells, produces IL-4 in response to SWAP, although the proportion of NK1.1+CD4+ T cells decreased after the infection (Fig. 1Gob). As STAT6-/- mice have impaired type 2 development of conventional T cells, NK1.1- T cells, but intact IL-4-producing NK1.1+CD4+ T cells (38, 39), we examined IL-4 production by hepatic lymphocytes from STAT6-/- mice in response to SWAP in the presence of Kupffer cells from infected wild-type mice. The profile of these cells in the liver is almost same in wild-type and STAT6-deficient mice (Fig. 5Goc). As shown in Fig. 5God, STAT6-deficient hepatic lymphocytes produced much less IL-4 than wild-type controls. Therefore, conventional T cells are a major source of IL-4 produced by SWAP-stimulated hepatic lymphocytes.

Differentiation into type 2 hepatic T cells by stimulation with SWAP

To confirm whether prompt IL-4 production by SWAP-activated hepatic T cells is due to their rapid differentiation into type 2 T cells, we examined their cytokine production profile in response to anti-CD3 challenge. Hepatic T cells from uninfected mice that had been stimulated with SWAP in the presence of infected mouse-derived Kupffer cells in vitro were stimulated with immobilized anti-CD3 mAb. As shown in Fig. 6Goa, they produced much more IL-4, IL-5, and IL-13, but much less IFN-{gamma}, than control hepatic T cells (Figs. 1Go and 2Go). When the hepatic T cells were incubated with SWAP in the presence of splenic macrophages from infected or uninfected mice or uninfected mouse-derived Kupffer cells instead of Kupffer cells from infected mice, the secondary culture revealed that hepatic T cells had the same cytokine production profile as that observed in the primary culture (data not shown). During the stimulation with SWAP presented by infected mouse-derived Kupffer cells, hepatic T cells rapidly differentiated into type 2 T cells.

Since Kupffer cells from the infected mice produced a considerable amount of IL-4, IL-6, and IL-10 in response to SWAP (Fig. 6Gob), and these cytokines have been reported to be involved in type 2 differentiation of T cells (30, 31, 32, 33, 34), we analyzed whether these cytokines derived from Kupffer cells contribute to the SWAP-induced prompt development of hepatic T cells into type 2 T cells. The amount of anti-IL-4 mAb used was 10-fold that which inhibited 10 ng/ml of IL-4 (data not shown). The amounts of anti-IL-10 and anti-IL-6 mAbs were dependent on the protocol. As shown in Fig. 6Gob, any treatment with anti-cytokine mAb did not remarkably down-regulate type 2 T cell differentiation of hepatic T cells determined by IL-4 production. These treatments also did not reduce the level of their type 2 differentiation measured by IL-13 or IL-5 production (data not shown). In addition, we examined the effects of anti-B7 mAbs on their quick type 2 differentiation, because B7.2 and B7.1 are relevant surface molecules for the development of Th2 and Th1 cells, respectively (40, 41). However, the development of hepatic T cells into type 2 T cells was not dramatically affected by these treatments (Fig. 6Gob).

Ultimate IL-4 production by hepatic lymphocytes induced by SWAP-activated Kupffer cells from infected mice in the egg deposit phase

Next, we investigated immunological properties of the hepatic immune system of S. mansoni-infected mice at the egg deposit phase. In the prepatent phase, hepatic T cells produced more IL-4 in response to SWAP than infected mouse-derived Kupffer cells ( Figs. 3–5GoGoGo). However, Kupffer cells began to produce a huge amount of IL-4 after the egg deposition stage (Table IGo and Fig. 3Goa), allowing us to investigate whether the main cell type producing IL-4 in response to SWAP changes from hepatic T cells to Kupffer cells after the egg deposition stage. As shown in Fig. 7Go, hepatic lymphocytes from the infected mice appeared to produce a tremendous amount of IL-4 upon stimulation with SWAP presented by Kupffer cells from infected mice at the egg deposit stage. However, this reflected the fact that >65% of the amount of IL-4 observed was produced by SWAP-stimulated infected mouse-derived Kupffer cells by themselves (10 wk; Table IGo and Fig. 7Go). Hepatic lymphocytes from any mice did not produce IL-4 under the same stimulation conditions, except for SWAP or Kupffer cells (data not shown). Thus, Kupffer cells acquired a much greater potential to produce IL-4 in response to SWAP in the egg deposit phase than in the prepatent phase, whereas hepatic T cells from mice in the egg deposit phase showed an equivalent level of IL-4 production as those in the prepatent phase.



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  		FIGURE 7. Ultimate IL-4 production by Kupffer cells from S. mansoni-infected mice (egg deposit stage) in response to SWAP. Kupffer cells were prepared from uninfected mice or S. mansoni-infected mice at 3 or 10 wk. They were incubated with (hatched, closed, and dotted columns) or without (open column) hepatic lymphocytes from uninfected mice (hatched column), infected mice at 3 wk (closed column), or infected mice at 10 wk (dotted column) in the presence of SWAP for 24 h. IL-4 in each supernatant was measured. The data are the mean ± SEM of triplicate samples from one experiment. The results shown are representative of three independent experiments. ND, not detected.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study clearly demonstrated for the first time that Kupffer cells have the capacity to produce both IL-4 and IL-13. To date, only T cells, particularly type 2 T cells and NK1.1+ CD4+ T cells, and myeloid cells, such as basophils, mast cells, and eosinophils, can produce IL-4 after the appropriate activation (29, 37, 42, 43). Kupffer cells also have capacity to produce IL-4 in response to the appropriate in vivo and in vitro stimulation (Table IGo and Figs. 3Go and 7Go). The mechanism by which Kupffer cells change to produce IL-4 and IL-13 is unclear, although a unique property of SWAP appears to play a critical role (Table IGo). A subpopulation of Kupffer cells predominantly producing IL-4 and IL-13 may proliferate after infection with S. mansoni. During the infection, Kupffer cells may gain a new signal transducing pathway to induce IL-4 and IL-13 production in response to SWAP. Kupffer cells may produce a new, unknown cytokine critical to the induction of IL-4 and IL-13 production. However, it is noted that these type 2-related cytokine-producing Kupffer cells are able to quickly change to produce IL-12 and IL-18 when stimulated with LPS (Table IGo), suggesting that S. mansoni infection does not delete the Kupffer cell population that preferentially produces IL-12 and IL-18 in response to LPS.

We demonstrated that SWAP, a crude mixture of worm-derived Ags, has potent activity to induce prompt IL-4 production and rapid type 2 differentiation in hepatic T cells isolated from healthy mice if presented by infected mouse-derived Kupffer cells ( Figs. 4–6GoGoGo). Macrophages from only S. mansoni-infected mice have the potential to present SWAP to T cells, while those from uninfected mice did not induce any IL-4 or IFN-{gamma} production by T cells in response to SWAP (Fig. 4Go). Moreover, Kupffer cells from infected mice have much greater ability to induce IL-4 production by lymphocytes than splenic macrophages from the same mice (Fig. 4Go). Several possibilities account for these differences. First, as adolescent worms reside in portal veins, Kupffer cells may receive particular and proper influences by interaction with adolescent worms and/or their products, which may not reach splenic macrophages. Second, as the liver is thoroughly the circulatory system directly connecting with the intestine, which is characterized to be almost equivalent to an outer environment, Kupffer cells may have more activity to respond to foreign molecules than splenic macrophages. Interestingly, hepatic lymphocytes from uninfected mice can produce much more IL-4 and IFN-{gamma} in response to immobilized anti-CD3 mAb than splenic lymphocytes from the same mice (Fig. 1Go). This may also reflect the anatomical condition of the liver, in that the hepatic immune system is endogenously activated to be more sensitive to exogenous stimulation than the splenic one. Furthermore, Kupffer cells from infected mice have the capacity to induce type 2 differentiation in hepatic T cells (Fig. 6Goa). This type 2 differentiation was not down-regulated by the neutralization of IL-4, IL-6, or IL-10 (Fig. 6Gob), which Kupffer cells produce in response to the same stimulation (Fig. 3Goa), indicating that type 2 differentiation-inducing activity of the Kupffer cells is independent of these cytokines. In addition, treatment of Kupffer cells with neutralizing anti-B7.2 did not result in inhibition of type 2 development of hepatic T cells, indicating that B7.2 is not involved in this type 2 differentiation. IL-13, presumably in collaboration with IL-4, produced by SWAP-stimulated Kupffer cells may largely contribute to induce type 2 T cell differentiation. Other unknown surface molecules that Kupffer cells become able to express after infection may contribute to induce this rapid type 2 T cell differentiation. Recently, it has been shown that monocyte chemoattractant protein-1, a member of the C-C-chemokines, up-regulates IL-4 production by spleen cells from SEA-sensitized mice in response to SEA, suggesting that our Kupffer cells might produce such a chemokine to help type 2 differentiation of hepatic T cells (44).

Many investigators have tried to identify the cell type that initiates differentiation of naive T cells into type 2 T cells. IL-4 is widely accepted as a prototype of Th2 cytokines (30, 31, 32). NK1.1+CD4+ T cells have been shown to produce IL-4 promptly in response to anti-CD3 mAb or anti-IgD Ab (29). However, NK1.1+CD4+ T cells are not required for S. mansoni infection-induced type 2 polarization in vivo, because ß2-microglobulin-deficient mice, lacking NK1.1+ T cells, showed type 2 polarization after the infection equal to that observed in wild-type mice (42). The liver contains many more NK1.1+CD4+ T cells than the spleen (12, 29), suggesting that NK1.1+CD4+ T cells might respond to SWAP stimulation with prompt IL-4 production. However, hepatic lymphocytes from uninfected STAT6-deficient mice, composed of NK1.1+CD4+ T cells with intact IL-4 production and conventional T cells impaired in type 2 differentiation (38, 39), produced much less IL-4 in response to SWAP presented by infected mouse-derived Kupffer cells (Fig. 5God). These results indicate that prompt IL-4 production in response to SWAP stimulation is mainly attributable to type 2 differentiated conventional T cells in the liver.

S. mansoni infection has been reported to result in the accumulation of IL-4-producing cells. Non-T, non-B, myeloid cells, including mast cells and basophils, have been demonstrated to be involved in type 2 polarization in the spleen (17, 18). Non-T, non-B cells produce IL-4 in response to IL-3 and/or SEA (17, 18). We observed that the Kupffer cell population mainly consisted of mononuclear phagocytes, not T cells, B cells, or mast cells (c-Kit+ cells), and produced IL-13 as well as IL-4 in response to SWAP (Table IGo and Figs. 3Go and 7Go). In addition, their capacity to produce IL-4 was tremendously enhanced as the infection was prolonged (Table IGo and Figs. 3Go and 7Go). This may not exclude the possibility that a minor population composed of contaminated myeloid cells such as basophils in the Kupffer cell fraction may contribute to this phenomenon. Recently, eosinophils have been demonstrated to be major source of Th2 cytokines in hepatic granuloma at egg deposit phase (45). The Kupffer cell fraction and hepatic lymphocyte fraction from the infected mice at the egg deposit phase (Fig. 7Go) were not obviously contaminated with eosinophils as determined by morphological study (data not shown). Therefore, we can only conclude that Kupffer cells, at least liver adherent cells, except T cells and mast cells, are able to promptly produce IL-4 in response to SWAP, which may in part contribute to the differentiation of hepatic T cells into type 2 T cells in vitro and possibly in vivo. We need further study to know whether SWAP-stimulated Kupffer cells or egg-elicited eosinophils are the major source of IL-4.

APCs are potent cells to determine the immune response to Ags. Macrophages produce IL-12 when stimulated with microbes or microbe products (9, 25). As previously reported, administration of heat-killed P. acnes, intracellular bacteria, render Kupffer cells to produce IL-12, which then induces type 1 differentiation in hepatic T cells (12). LPS also stimulates Kupffer cells to produce IL-12 as well as other proinflammatory cytokines directly (25). Dendritic cells have been shown to produce IL-12 when presenting Ag to naive CD4+ T cells (46, 47). In the case of S. mansoni infection, either uninfected or infected mouse-derived Kupffer cells preferentially produce both IL-6 and IL-10, but not IL-12 or IL-18, in response to SWAP. IL-12 is an essential factor for type 1 differentiation, and IL-18 markedly enhances it in collaboration with IL-12 (6, 7, 8, 9, 10, 11). IL-6 and IL-10 play some role in direction of T cells to type 2 cells (33, 34) and may exert their actions much more efficiently in the absence of IL-12 and/or IL-18 than in their presence. However, these characteristics are still inadequate to induce differentiation of naive T cells into type 2 T cells (Fig. 6Go). Rather, it may be important that infected mice-derived Kupffer cells become able to produce IL-4 and IL-13 in response to SWAP (Fig. 4Go and Table IGo). In contrast, S. mansoni-infected mouse-derived splenic macrophages did not produce IL-4 (Fig. 4Go) or IL-13 (data not shown) in response to the same stimulation. Recently, dendritic cells, professional APCs, were functionally divided into two populations according to their ability to induce Th1 and Th2 differentiation, and D2 cells help Th2 differentiation (48). Like dendritic cells, Kupffer cells may be divided into two populations, and Kupffer cells from infected mice may belong to a type 2 T cell differentiation-inducing subset. Thus, Kupffer cells acquire IL-4- and IL-13-producing activity after infection with S. mansoni, presumably due to interaction with adolescent worm products, possibly leading to the unique feature of the S. mansoni-infected liver.

The infected mouse-derived Kupffer cells do not solely contribute to the prompt type 2 response during infection. Because of the migration pattern exhibited by the parasite, skin draining lymph nodes and lungs could contribute to a rapid Th2 response. Wilson et al. demonstrated that irradiated cercariae are able to promote a protective Th1 response, while normal unattenuated parasites elicited higher IL-4 and IL-5 expression upon both primary and secondary stimulations (49). These findings were documented in skin draining lymph nodes, very early after exposure to the parasites. Similar findings were reported in a study by Wynn et al., in which the cytokine response to both irradiated and normal parasites was studied in the lung 6–28 days after exposure to parasites (50). Here, as in the study by Wilson et al., normal parasites stimulated a rapid type 2 response in the lung, which was obvious as early as 6–10 days after the animals were first exposed to the parasites. Several studies have shown convincingly that parasites can sensitize animals for granuloma formation and Th2 response (51, 52, 53).

In summary, the liver displayed a unique immune response to S. mansoni infection, showing straightforward type 2 deviation after S. mansoni infection. Hepatic lymphocytes derived from uninfected mice promptly differentiated into type 2 T cells in response to stimulation with SWAP presented by infected mouse-derived Kupffer cells. Kupffer cells themselves became able to produce IL-4 and IL-13 in response to SWAP after S. mansoni infection, and the amount of IL-4 produced was elevated as the infection was prolonged, particularly after deposition of eggs. On the basis of these unique responses of hepatic lymphocytes as well as Kupffer cells to SWAP, the liver T cells may promptly direct to type 2 polarization after infection. We are now investigating the precise mechanism by which S. mansoni-infected mouse-derived Kupffer cells induce type 2 differentiation in hepatic T cells.


   Acknowledgments
 
We thank Dr. Tomohiro Yoshimoto for enthusiastic discussion. We are grateful to Mss. Aki Suzuki and Shizue Futatsugi for excellent technical assistance.


   Footnotes
 
1 This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (351,366); a Hitec Research Center Grant from the Ministry of Education, Science, Culture, and Sports; and the Osaka Foundation for Promotion of Clinical Immunology, Japan. Back

2 Current address: Laboratory of Immunology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892-1892 Back

3 Address correspondence and reprint requests to Dr. Kenji Nakanishi, Department of Immunology and Medical Zoology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan. E-mail address: Back

4 Abbreviations used in this paper: SEA, S. mansoni egg Ag; SWAP, soluble worm Ag preparation. Back

Received for publication May 25, 1999. Accepted for publication October 6, 1999.


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